For all intracellular trafficking events it is indispensable, that two membranes fuse with each other resulting in lipid and content mixing. This fundamental process is supposed to be catalyzed by specific proteins termed SNAREs (soluble NSF attachment protein receptors). Their sequence and structure is conserved in all eukaryotic systems, beginning with yeast and ending in humans. Since vesicle fusion is involved in many essential functions like synaptic transmission, hormone secretion and endocytosis, detailed knowledge about the molecular mechanism, how this reaction is catalyzed and what proteins besides of SNAREs are needed to fulfill this function will have deep impact on many disease-relevant topics. In order to unravel the precise fusion mechanism the yeast vacuolar fusion system is employed. Yeast vacuoles are purified in a large-scale isolation procedure and reconstituted in an in vitro system. Fusion efficiency of vacuoles can be easily measured by using two different strains, one lacking the vacuolar alkaline phosphatase PHO8, the other lacking the vacuolar protease PEP4. PHO8 is present as an inactive pro-enzyme, which needs to be cleaved in order to be active. After fusion has occurred, PEP4 gains access to the immature PHO8 enzyme, cleaves it and therefore activates it. Phosphatase activity can easily measure spectrophotometrically using a well-known assay. Vacuoles can be isolated in large quantities facilitating biochemical investigations. Since this system is the only existing possibility to address topological issues for membrane fusion in a physiological context in terms of how proteins interact in trans (between two fusing membranes), we reinvestigated a current dogma entitled as topological restriction model. We took advantage of a certain property of yeast vacuolar fusion, namely that fusion rate is relatively slow and therefore can easily dissected in different stages. We have discovered an unexpected post-priming cis-SNARE complex, which we will characterize further in terms of how its stability is controlled and what other proteins are needed to stabilize this complex. Aim 1 will address this issue. Furthermore, we have discovered another topological trans-interaction of SNARE proteins, not predicted by the current model by using differently tagged SNAREs on the two fusing vacuoles. The fusion relevance of this trans-interaction was investigated by employing different combinations of vacuoles containing specifically inactivated SNAREs on their surface. We will continue to generate SNARE mutants to further confirm the physiological relevance of the discovered trans-SNARE topology (Aim2). We also started to elucidate, what specific function additional factors like the SEC1/MUNC18 related HOPS-complex and the V0 part of the V-ATPase have for the membrane fusion process. We have characterized V0 mutants, which are unable to fuse but have not lost their ability to pump protons. Aim 3 of this proposal addresses this topic. PUBLIC HEALTH RELEVANCE: SNARE (soluble NSF attachment protein receptors)-mediated intracellular fusion is essential for synaptic transmission, hormone secretion and endocytosis. Many diseases in synaptic transmission are linked to a disorder of SNARE-function and their associated proteins. Also some sorts of decreased insulin secretion are caused by insufficient SNARE-function resulting in Diabetes-like phenotypes.